Matrix with immunomodulating activity

ABSTRACT

The invention claims an iscom matrix which is not a lipid vesicle comprising at least one lipid and at least one saponim but no intentional antigenic determinants and optionally also adjuvants for use as an immunomodulating agent, medicines, vaccines, kits containing the matrix and new saponins, and a process for preparing the new saponins. The invention also concerns a process for preparing the matrix characterized in that at least one sterol is solubilized in a solvent or detergent, the saponin or saponins are added, the other adjuvants and lipids are optionally also added, whereafter the organic solvent or the detergent may be removed for example with dialysis, ultra filtration, gel filtration or electrophoresis. The sterol and saponin might also be solubilized in the lipids and/or adjuvants.

This is a continuing application of Ser. No. 07/251,576 which was filedon Sep. 30, 1988, and now abandoned.

The present invention concerns an iscom matrix comprising at least onelipid and at least one saponin with immunomodulating effect, a processfor preparing the matrix, a vaccine and a kit comprising the same andnew saponins for incorporation in the matrix and a process for preparingthe new saponins.

Many microbial and vital antigens can be produced by modern techniquestoday. Their full promise in vaccines will however not be realizedunless they are administered along with an effective adjuvant, an agentthat increases antibody and/or cell-mediated immune responses.

The only adjuvants currently authorized for human use in most countriesare aluminum hydroxide and aluminum phosphate which have been used formany years to increase antibody responses to e.g. diphtheria and tetanustoxoids. Although these adjuvants are sufficient for many vaccines,studies have shown that other adjuvants, e.g. Freund's complete adjuvant(FCA), and Quil A often are more efficacious in eliciting antibodyresponse and cell-mediated immunity in experimental animals. In fact,they are frequently required for protection. However. FCA producesgranulomas at injection sites, which makes them unacceptable for humanand veterinary vaccines. In fact, even aluminum hydroxide may give riseto reactions in form of granuloma at the injection site. For thesereasons, many attempts are made to develop adjuvants with the efficacyof FCA but without undesirable side effects.

In Morein's EPC Patent Applications Nos. 83850273.0 and 85850326.1,there are described immunogenic complexes between antigenic determinantswith hydrophobic regions and glycosides, among them triterpensaponinsand especially Quil A, so called iscom complexes. In such an iscom, theamount of Quil A can be about 10-100 times lower and produce the sameantigenic effect as when Quil A in free form is mixed with the antigen.

European Patent Application 87200035.1 indicates that the presence ofantigen is not necessary for formation of the basic iscom structure,this being possible to form from a sterol, such as cholesterol, aphospholipid, such as phosphatidylethanolamine, and a glycoside such asQuil A.

It has now been discovered that a phospholipid is not needed for thepreparation of the basic iscom structure including no antigen. Instead asterol, such as cholesterol in conjunction with a glycoside such as QuilA are the essential structural components assembled into a complexresembling the typical cage-like iscom structure, so called matrix. Ithas also turned out that the matrix has immunomodulating effects such asadjuvant or immunosuppressive effect.

The present invention concerns a complex between at least one lipid suchas a sterol, preferably cholesterol, and one or more saponins, such astriterpensaponins, especially Quil A or subcomponents thereof which isnot a lipid vesicle without any intentional antigens or antigenicdeterminants for use as an immunomodulating agent. Thus, there is notintegrated any antigenic component as is done in an iscom. This matrixhas adjuvant effect and can be used mixed together with one or moreantigens preferably in multimeric form.

In this iscom matrix there is also possible to integrate other adjuvantswith hydrophobic regions. Addition of other lipids may be required tofacilitate the inclusion of other adjuvants. Thus the present inventionalso concerns a complex containing lipids and adjuvants, other thancholesterol and saponins. Such complexes contains the matrix consistingof cholesterol and saponin, preferably Quil A or subcomponents thereof,one or more other adjuvants and one or more lipids other thancholesterol. These are preferably not lipid vesicles or liposomes andhave a very special structure in electron microscopy.

Liposomes have been described in the literature and their generalstructure is well known to biological research workers. Liposomes arevesicles comprising one or more series of lipid layers formingonion-like structures spaced one from another by aqueous material.

The matrix can be injected in an animal or human being as a mixture withthe antigen in multimeric form. Alternatively the matrix and the antigencan be injected separately. In this case the best results are obtainedif the adjuvant matrix and the the antigen are injected in regions whichare drained into the same lymphatic gland. When the adjuvant ispresented in multimeric form in a matrix according to the invention thedose of adjuvant may be lowered as compared with when the adjuvant isinjected separately in monomeric form or in an undefined form. Thisimplies that toxic side effects caused by adjuvants when usedconventionally, i.e. when they are injected alone as such, can belowered or avoided. The dose of adjuvant can, however, not be lowered asmuch as is done in the iscom complexes according to the above mentionedpatent applications.

When an adjuvant is used in a matrix according to the invention, theantigen is not integrated in the same particle as the adjuvant as isdone in an iscom particle according to the above mentioned EPC PatentApplications. This implies that one can use antigens without amphiphaticproperties or antigens which can not be forced to expose hydrophobicregions. As an example it can be mentioned that some viruses do not haveamphiphatic proteins, e.g. picornavirus, adenovirus or parvovirus, butthey have a form of submicroscopic particle with the antigen presentedin several copies, i.e. as multimers.

For such viruses it is more practical to inject them together with thenew adjuvant complex than to couple hydrophobic groups to them or createhydrophobic groups by other means (e.g. partial denaturation) andintegrate them into an iscom particle.

Typically, the present matrix contains sterol, preferably cholesterol,and one or more saponins in a molar ratio of about 1 to 1 or in a weightratio of about 1 to 5. The complexes have an open sperical structureconsisting of circular subunits or parts of the spherical structurerevealed by electron microscopy. They have a sedimentation coefficientof about 20 S.

When other adjuvants are integrated, the lipid-adjuvant-matrix typicallycontains sterol and saponin in a molar ratio of about 1:1 and the otheradjuvants and lipids together make up to about 1 molar. For such amatrix the molar ratio of sterol; saponin; other adjuvant and lipids isabout 1:1:1. Thus the molar ratio of sterol; saponin; other adjuvant andother lipids is 1:1:0,1-1; 0,1-1, i.e. additional lipid or adjuvant maybe present in the matrix until its molar ratio (or the sum of theirmolar ratios) is a half that of the saponin and sterol present.

The structure as revealed by electron microscopy is the same as foriscom and matrix (see FIG. 1).

The sedimentation coefficient, being dependent on the density ofmaterial incorporated into the matrix, is about 12-22 S for matricescontaining cholesterol, saponin, other adjuvants and lipids.

The saponins can be any saponin with hydrophobic regions such as thosedescribed in R Tschesche and Wulf, Chemie und Biologic der Saponine inFortschritte der Chemie Organischer Naturstoffe, published by W Herz, H,Grisebach, G W Kirby, Vol 30 (1973), especially the strongly polarsaponins, primarily the polar triterpensaponins such as the polar acidicbisdesmosides, e.g. saponin extract from Quillsjabark Araloside A,Chikosetsusaponin IV, Calendula-Glycoside C, Chikosetsusaponin V,Achyranthes-Saponin B. Calendula-Glycoside A, Araloside B, Araloside C,Putranjia-Saponin III, Bersamasaponiside, Putrajia-Saponin IV,Trichoside A, Trichoside B, Saponaside A, Trichoside C, Gypsoside.Nutanoside, Dianthoside C, Saponaside D, preferably aescine fromAesculus hippocastanum (T Part and W Winkler: Das therapeutisch wirksamePrinzip der Rosskastanie (Aesculus hippocastanum), Arzneimittelforschung10(4), 273-275 (1960) or sapoalbin from Gyposophilla struthium (RVochten, P Joos and R Ruyssen: Physico-chemical properties of sapoalbinand their relation to the foam stability, J Pharm Belg 42, 213-226(1968), especially saponin extract from Quillaja saponaria Molina,primarily the DQ-extract which is produced according to K Dalsgaard:Saponin Adjuvants, Bull Off Int Epiz 77 (7-8), 1289-1295 (1972) and QuilA which is produced according to K Dalsgaard: Saponin Adjuvants III,Archiv fur die Gesamte Virusforschung 44, 243-254 (1974). Quil A andsubfragments thereof are preferred, especially the fragments B2, B3 andB4B described below.

The present invention also provides new glycosylated triterpenoidsaponins derived from Quillaja Saponaria Molina of Beta Amytin type with8-11 carbohydrate moieties which have the following characteristics:

a) Substance B2 has a molecular weight of 1988, a carbon 13 nuclearmagnetic resonance (NMR) spectrum as indicated in FIGS. 5A and 6A and aproton NMR spectrum as shown in FIGS. 11A and 12A.

b) Substance B3 has a molecular weight of 2150 and has a carbon 13 NMRspectrum as shown in FIGS. 5B and 6B, and a proton NMR spectrum as shownin FIGS. 11B and 12B.

c) Substance B4B has a molecular weight of 1862, a carbon 13 NMRspectrum as shown in FIGS. 5C and 6C, and a proton NMR structure asshown in FIGS. 11C and 12C.

Compounds B2 and B3 have adjuvant activity in their own right. Thepresent invention also relates therefore to the use of these compoundsas adjuvants. Compound B4B is of use in the preparation of an iscommatrix. B2 and B3 having adjuvant activity can be included in thematrix.

Matrix can be produced from a sterol such as cholesterol and the saponinB4B. Such a matrix does not seem to have any potent adjuvant activity.In order to potentiate the adjuvant activity in this matrix, it ispossible and even preferable to integrate the saponins B2 and/or B3and/or any other substance with adjuvant effect and with hydrophobicgroups. If the adjuvants do not contain any hydrophobic groups suchgroups might be coupled to them by use of known chemical methods. Ifother adjuvants than B2 or B3 are to be integrated, there are preferablyincorporated further lipids as listed hereinafter.

In the sterol-B4B matrix, it is also possible to integrateimmunosuppressive substances containing hydrophobic groups or to whichsuch groups have been coupled.

It is also possible to use the sterol-B4B matrix as an immunomodulatingagent in mixture with adjuvants, immunosuppressive substances orantigens or mixtures thereof.

As immunodulating agents are considered substances that enhance,suppress or change the immune system such as adjuvants, suppressors,interleukins, interferons or other cytokins.

The invention preferably concerns an matrix containing a sterol,especially cholesterol, B4B and either of B2 and B3 or both. When matrixis prepared from cholesterol and Quil A, it comprises B2, B3 and B4B.

The matrixes can be produced by solubilizing at least one sterol in asolvent, adding the saponin or saponins, and possibly the otheradjuvants and lipids, whereafter the solvent might be removed and thematrix transformed into a solution where its components are not soluble,e.g. a water solution. This can be done with gel filtration, ultrafiltration, dialysis or electrophores. The matrices may then be purifiedfrom excess of sterol and Quil A e.g. by centrifugation through adensity gradient, or gel filtration. As solvent there might be usedwater or the solubilizing agents or detergents mentioned below.

The only limiting factor for matrix formation to take place is the timeneeded in different physico-chemical environments, the major ratelimiting factor being the poor solubility of the sterol, e.g.cholesterol, in water, in which the matrix forming saponins are freelysoluble.

Thus it has been shown that with Quil A and cholesterol even in solidphase matrix-like formation takes place after a relatively long time,e.g. about 1 month. Cholesterol must be brought into contact with Quil Aor its purified components. If the cholesterol is brought into colloidalwater suspension through treatment by ultrasonication and treatment byultra-turrax, matrix is formed with Quil A after about 12 hours.

Consequently, any other substance such as a detergent added to thewater, and which will increase the solubility of cholesterol in theaqueous medium, will decrease the time needed for the formation ofmatrix. It is thus possible to produce a matrix from cholesterol, waterand Quil A or the subcomponents thereof, if the cholesterol is broughtto a colloidal form. It is, however, more practical to add a detergentor a solvent.

Preferably the saponins are used from a concentration of at least theircritical micelle formation concentration (CMC). For Quil A this impliesa concentration of at least 0.03% by weight.

As solubilizing agent there can be used detergents such as non-ionic,ionic i.e. cationic or anionic or Zwitter-ionic detergent such asZwittergent or detergent based on gallic acid which is used in excess.Typical examples of suitable non-ionic detergents areN-alkanoyl-N-alkyl-glucamines, polyglycol esters and polyglycol etherswith aliphatic or aralylphatic acids and alcohols. Examples of these arealkylpolyoxyethylene ethers with the general formula C_(n) H_(2n+1)(OCH₂ CH₂)_(x) OH, shortened to C_(n) E_(x) ; alkyl-phenylpolyoxyethylene ethers containing a phenyl ring between the alkyl groupand the polyoxyethylene chain, abbreviated C_(n) φE_(x), TritonX-100=tert.-C₈ E₉.6 (octylphenolether of polyethylene oxide),acylpolyoxyethylene esters: acylpolyoxyethylene sorbitane esters,abbreviated C_(n) sorbitane E_(x), e.g. Tween 20, Tween 80,β-D-alkylglucosides, e.g. β-D-octylglucoside. Typical examples ofsuitable ionic detergents are gallic acid detergents such as e.g. cholicacid, desoxycholate, cholate and CTAB (cetyltriammonium bromide). Evenconjugated detergents such as e.g. taurodeoxyoholate, glycodeoxycholateand glycocholate can be used. Other possible solubilizing agents arelysolecithin and synthetic lysophosphoilipids. Even mixtures of theabove-mentioned detergents can be used. When using the dialysis methodthe detergents should be dialysable in not too long time.

Some surface active substances greatly facilitate matrix formation.These include the intrinsic biological membrane lipids with a polar headgroup and a non-polar aliphatic chain e.g. phosphatidyl choline(negatively charged) and phosphatidyl ethanolamine (positively charged).

Solubilizing can also be performed with alcohols, organic solvents orsmall amphiphatic molecules such as heptane-1,2,3-triol,hexane-1,2,3-triol or caotrophic substances, acetic acid, such astrifluoro-acetic acid, trichloro-acetic acid, urea or quanidinehydrochloride.

Preferably to be used are ethyl alcohol, dioxane, ether, chloroform,acetone, benzene, acetic acid, carbon disulphid, MEGA-10(N-decanoyl-N-methyl glucamine) and β-octylglucoside.

Various yields of matrix can be obtained with these substances, and theoverall picture is that more matrix is formed the higher theconcentration of the detergent is in the system.

It is technically possible to produce, purify, and sterilize matrix inany of the systems described. Therefore the adjuvant active technicalpreparations of matrix may contain solubilizing agents if their chemicalnature and their concentration is acceptable in the final product, e.g.for vaccine purposes. However, in many cases, it will be necessary toremove the solubilizing agent from the matrix by dialysis,ultrafiltration or column chromatographic techniques. It is evenpossible to dilute the preparation until an allowed concentration of agiven solubilizing agent or detergent is reached. The preparation isdiluted with water or a physiologically acceptable solution preferablyto a concentration below the CMC for the solubilizing agent or detergentin the system (the preparation) used.

The solubilizing agent might be incorporated in the matrix in a molarratio of sterol saponin: further lipid adjuvants or solubilizing agent1:1:1, i.e. molar ratio of the sum of lipid, adjuvants and solubilizingagent is up to half the molar that of saponin and sterol.

The solubilizing agent might alternatively be left mixed with the iscommatrix.

In order to be integrated the solubilizing agent and otherimmunomodulating components, should have at least one hydrophobicregion. If not present such hydrophobic regions can be coupled to thecomponents before the matrix is made.

Examples of adjuvants that can be incorporated in iscom matrix are anyadjuvant, natural or synthetic, with desired immunomodulatory effect,e.g. muramyl dipeptide (MDP)-derivatives, such as fatty acid substitutedMDP, threonyl analogs of MPD; amphipatic copolymers aliphatic aminessuch as avridine or DDA, poly anions such as Dextran sulphate,lipopolysaccarides such as saponins (other than Quil A). ("Futureprospects for vaccine adjuvants", Warren, H. S. (1988) CRC Crit. Rev.Immunol. 8:2, 83-101; "Characterization of a nontoxic monophosphoryllipid A", (1987) Johnson, A. G. et al, Rev. Infect. Dis. 9:5, 5512-5516;"Developmental status of synthetic immunomodulators", Berendt, M. J. etal (1985), Year Immunol. 193-201: "Immunopotentiating conjugates",Stewart-Tull, D. E., Vaccine, 85, 3:1, 40-44).

These four references are hereby incorporated as references.

The following Zwitterionic, neutral, positive and negative detergentsare examples of detergents that have immunomodulating, especiallyadjuvant activity:

Nonionic block polymer surfactants containing hydrophilicpolyoxyethylene (POE) and hydrophobic polyoxypropylene (POP) thatdiffered in mol weight percentage of POE and mode of linkage POP to POE(BASF Wyandotte Corp.), such as L72, L81, L92, pluronic L101, L121, 2531and 31R1: octablocks T1501; B-D-octylglucosid; cationic surfactants suchas dimethyldioctadecylammonium bromide (DDA), octadecylamine (OCT), andcetyltrimethylammonium bromide (CTAB); maltostose tetrapalmirate,trehalose monomycolate, trehalose dibehenylbehenate: zwittergentdetergents (N-alkyl-N,N-dimethyl-ammonio-3-propanesulphonate) Z3-8,Z3-10, Z3-12, Z3-14, Z3-16, obtained from Calbiochem (La Jolla, Calif.,USA); Z3-18 obtained from Serra (Heidelberg, FRG), Myrj 45, Brij 52,Brij 58 (also from Serva), and dioctylsulphosuccinate and Tween 20,Tween 80, Triton X-100 and sodium deoxycholate.

These detergents can be used as both detergents and adjuvants and beincorporated in the iscom matrix.

The following are examples of immunosuppressive agents that can beincorporated in a sterol (preferably cholesterol) B4B matrix:cyclosporin A, diodecyl dimethyl ammonium bromide, cationic single chainamphiphiles with more than 10 carbon atoms and preferably more than 15carbon atoms, double chain amphiphiles with up to 14 carbon atoms,preferably up to 12 carbon atoms.

In the case a desired adjuvant or immunosuppressive agent do not havesuitable hydrophobic properties, it has to be modified to comprise ahydrophobic domain for incorporation into the matrix.

The hydrophobic group that can be coupled to non-hydrophobic adjuvantsare straight, branched, saturated or unsaturated aliphatic chains having1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24 and 25 carbon atoms, such as lipids, preferably 6, 7 and8 carbon atoms: small peptides with 1, 2, 3, 4 or 5 amino acids,preferably 2, 3, 4, selected from Trp, Ile, Phe, Pro, Tyr, Leu, Var,especially Tyr; choline acid, ursodesoxycholine acid or cholesterolderivatives.

These hydrophobic groups must be bonded to a group that can be coupledto the non-hydrophobic protein such as carboxyl-, amino-, disulphide-,hydroxyl-, sulphydryl- and carbonyl group, such as aldehyde groups.

As hydrophobic groups that can be coupled are selected preferablycarboxyl, aldehyde, amino, hydroxyl, and disulphide derivatives ofmethane, ethane, propane, butane, hexane, heptane, octane and peptidescontaining Cys, Asp, Glu, Lys, preferably octanal and Tyr.Tyr.Tyr-Cys,-Asp or -Glu. The hydrophobic groups with a group that can be coupledmust be dissolved in water with the aid of for example the solubilizingagents and detergents mentioned above or hydrochloric acid, acetic acid,67% by volume acetic acid, caustic liquor, ammonia, depending on whatsubstance is to be dissolved. pH is then adjusted to the neutraldirection without the substance precipitating: here it is to make surethat there is not obtained a pH-value that denaturates the protein towhich the hydrophobic group is to be coupled.

Hydrophobic groups with a carboxyl group as coupling molecule can becoupled to the adjuvants through water-soluble carbodiimides orcomposite anhydrides. In the first case the carboxyl group is activatedat pH 5 with carbodiimide and mixed with the protein dissolved in bufferpH 8 with a high phosphate content. In the latter case the carboxycompound is reacted with isobutylchloroformate in the presence oftrimethylamine in dioxane or acetonitrile, and the resulting anhydrideis added to the protein at pH 8 to 9. It is also possible to convert thecarboxyl group with hydrazine to hydrazide which together with aldehydesand ketones in periodate-oxidized sugar units in the protein giveshydrazone bonds.

The amino groups with nitrous acid can at low temperature be convertedto diazonium salts, which gives azo bonds with Tyr, His and Lys.

The hydroxyl groups with succinic anhydride can be converted tohemisuccinate derivatives which can be coupled as carboxyl groups.

Aldehyde groups can be reacted with amino groups in the protein to aSchiff's base.

Several coupling groups and methods are described in Journal ofImmunological Methods, 59 (1983) 129-143, 289-299, Methods in EnzymoloyVol 93 pp 280-33, and in Analytical Biochemistry 116, 402-407 (1981)which are here incorporated as references.

The lipids other than sterol can be fats or fat resembling substancessuch as triglycerides or mixed triglycerides containing fatty acids withup to 50 carbon acids such as saturated fatty acids with 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29 and 30 carbon atoms e.g. burytic acid, caprole acid, caprylicacid, captic acid, lauric acid, myristic acid, palmitic acid, stearicacid, arachidic acid, behenic acid, lignoceric acid, or unsaturatedfatty acids with up to 30 carbon atoms, such as hexadecene acid, oleicacid, linoleic acid, linolenic acid, arachidonic acid: hydroxy-fattyacids such as 9,10-dihydroxy stearic acid, unsaturated hydroxy fattyacids such as castor oil, branched fatty acids: glycerol ethers, waxesi.e. esters between higher fatty acids and monohydric alcohols:phospholipides such as derivatives of glycerol phosphates such asderivatives of phosphatidic acids i.e. lecithin, cephalin, inositolphosphatides, spingosine derivatives with 14, 15, 16, 17, 18, 19 and 20carbon atoms: glycolipids isoprenoids, sulpholipids, carotenoids,steroids, sterols, cholestanol, caprostanol, phytosterols, e.g.stigmasterol, sitosterol, mycosterols, e.g. ergosterol, bile acids e.g.cholic acid, deoxycholic acid, chenodeoxycholic acid, litocholic acid,steroid glycosides, esters of vitamins A, or mixtures thereof.

These and other useful lipids are described in: Lipid biochemistry andintroduction, Ed. M. I. Gurr, A. I. James, 1980, Chapman and Hall,London, New York, University Press Cambridge, which hereby isincorporated as a reference.

Preferably cholesterol phosphatidyleholine, liposomes or intralipid®(Oleum soya fractionate 200 g, Lechitinum fractionate vitello ovi 12 g,glycerol 22.5 g, and H₂ O up to 1 liter) are used.

The lipids can be added at any stage in the process, preferably beforethe addition of the saponin but lipids could also be added after thesaponin.

The matrix is best produced by the dialysis method as follows.

Cholesterol dissolved in 20% MEGA-10 or any other suitable detergent,preferably a detergent that can be removed by dialysis, e.g.β-octylglucoside, (in H₂ O or a suitable buffer) is mixed with 5 timesas much Quil A (solid or dissolved in water or a suitable buffer, e.g.PBS). The mixture is dialysed extensively against PBS, first over nightat room temperature (because MEGA-10 will precipitate at +4° C.), thenat +4° C. The matrixes are purified from excess Quil A and cholesterolby pelleting through e.g. 30% (w/w) sucrose (e.g., a TST 41.13 rotor 18h, 39.000 rpm, 10° C.). The pelleted matrixes are dissolved in PBS (orany other suitable buffer) and the concentration adjusted to 1 mg/ml).

The present matrix can be used as an immunomodulating substance. It canbe used as a potentiating agent for an immunosuppressive substance or anadjuvant, either mixed therewith or integrated in the matrix.

A matrix containing a sterol such as cholesterol, saponins, adjuvantsand optionally further lipids can be used as an adjuvant. It can be usedfor potentiating the antigenic effect of any antigen or antigenicdeterminants from any pathogenic organism or any fragments or subunitsof, or derived from these. Thus it can be used as an adjuvant for thoseantigens that are integrated in an iscom. Such antigens are mentioned inthe EPC-patent applications 83850273.0 and 85850326.1, which are herebyincorporated as references. Thus the matrix can be used as adjuvantstogether with antigens or antigenic determinants derived from viruseswith or without envelope, bacteria, protozoa, mycoplasmas, helminths,mollusca or together with such whole organisms. The antigenes orantigenic determinants might further be hormones, enzymes, carbohydratesand carbohydrate-containing structures such as lipopolysaccharides,peptides or proteins or recombinants thereof.

The present invention thus also covers human or veterinary medicine,characterized in that it comprises at least one matrix and one or moreantigenic or immunosuppressive substances and a pharmaceuticallyacceptable vehicle in mixture or in separate compartments.

The invention also concerns a vaccine comprising an matrix, one or moreantigens and a pharmaceutically acceptable vehicle.

Further the invention concerns a kit comprising such a medicine orvaccine.

In some medicines or vaccines the detergent used when preparing thematrix can be present if the detergent is allowed for the product inquestion.

The effect of the new adjuvant complex according to the invention willnow be described in immunostimulating experiments.

1. Comparison between the immunogenic effects from antigens presented asiscoms, micelles or micellas plus the new matrix.

Mice were immunized with envelope protein from influenza virus in theform of iscom complex, micelles and micelles together with the newcomplex according to the invention (so called matrix). The immuneresponse was evaluated by measuring the antibodies with ELISA technique15, 30, 44 and 50 days after injection. The following injections weremade:

1. 5 μg Micelle+0.1 μg matrix were mixed and injected in the leftforeleg.

2. 5 μg Micelle+0.1 μg matrix injected separately in the right and leftforeleg respectively.

3. 5 μg Micelle

4. 5 μg iscom prepared according to EPC 83850273.0

                  TABLE 1                                                         ______________________________________                                        DAY      1        2          3      4                                         ______________________________________                                        15       23.700 ±                                                                            12.900 ±                                                                              18.900 ±                                                                          41.600 ±                                        9.500    14.500     9.500  1.000                                     30       30.800 ±                                                                            8.800 ± 9.800 ±                                                                           80.700 ±                                        10.500   7.100      3.600  21.700                                    44       30.000 ±                                                                            32.600 ±                                                                              17.900 ±                                                                          129.00 ±                                        17.600   17.300     4.200  78.400                                    50       309.300 ±                                                                           136.700    87.600 ±                                                                          880.430 ±                                       89.000   103.700    18.200 295.500                                   ______________________________________                                    

No side effects in the form of local reactions were noted.

From this experiment one can conclude that envelope protein frominfluenza in the form of iscom or micelles plus matrix gives the highestantibody titres. Matrix can be presented in a very low dose and stillhave adjuvant effect. In order to get an adjuvant effect in mice, Quil Ain free form is required in a dose a 100 times the dose of matrix, 10μg. With that dose Quil A begins to give local side reactions. In orderfor matrix to have an obvious adjuvant effect the antigen in multimericform should be injected in the same region e.g. leg as the matrix, i.e.the injected adjuvant matrix complex and antigen should be presented ina region, that is drained to the same lymphatic gland.

2. Comparison between the immunogenic effects from envelope protein frominfluenza in the from of iscom or micelle with or without matrix ordiphtheria toxoid (DT).

Mice were injected with envelope protein in the following forms:

1. 5 μg Iscom+5 μg DT

2. 5 μg Iscom

3. 5 μg micelle

4. 5 μg micelle+0.1 μg matrix

The antibody response in envelope proteins was estimated in the serumwith ELISA-technique. The following results were obtained:

                  TABLE 2                                                         ______________________________________                                        DAY     1         2           3     4                                         ______________________________________                                        15       52.800.sup.1)                                                                           48.300     8.400 29.000                                    30      119.202   155.567     22.107                                                                              87.000                                    50      110.600   136.200     33.400                                                                              96.500                                    65      1.691.000 3.783.000   283.300                                                                             1.149.000                                 80      562.800   2.529.000   512.300                                                                             976.500                                   ______________________________________                                         .sup.1) ELISA-titer where the last dilution gives a significant positive      value at 450 nm.                                                         

No visible side effects in form of local reactions could be noted.

One can conclude that envelope protein from influenza virus in the formof iscom or micelles plus the adjuvant complex (matrix) according to theinvention gives the highest antibody titres. The dose of matrix can bekept very low, i.e. 0.1 μg, and still has a notable adjuvant effect.

3. Comparison between the immunogenic effects from diphtheria toxoid(DT) in monomeric form, monomeric DT+iscom containing envelope proteinfrom influenza virus, monomeric DT in mixture with Quil A andcholesterol and monomeric DT+adjuvant complex (matrix) according to theinvention.

In this experiment diphtheria toxoid is used as a model antigen inmonomeric form.

Mice were injected with diphtheria toxoid in the following forms:

1. 5 μg DT (diphtheria toxoid)

2. 5 μg DT+5 μg iscom

3. 5 μg DT+0.5 μg Quil A+0.1 μg CL (cholesterol)

4. 5 μg DT+0.1 μg matrix

                  TABLE 3                                                         ______________________________________                                        DAY       1      2          3    4                                            ______________________________________                                        15        ≦30                                                                           ≦30 ≦30                                                                         ≦30                                   30        ≦30                                                                           ≦30 ≦30                                                                         ≦30                                   50        ≦30                                                                           ≦30 ≦30                                                                         ≦30                                   65        ≦30                                                                           90         ≦30                                                                         10.000                                       80        ≦30                                                                           60         90   1.10                                         ______________________________________                                    

The immungenic response to DT is low in all the groups. The best resultis obtained with mice immunized with diphtheria toxoid plus matrixaccording to the invention.

From the experiments above one can conclude that the best results areobtained when the matrix according to the invention is used togetherwith the antigen in multimeric form. The matrix according to theinvention has thus proved to give very good results as adjuvant comparedwith e.g. Quil A in free form. Thus it is worth noting that Quil A iseffective as adjuvant in free form in doses such as 10 μg for mice, 50μg for guinea-pigs and 1 mg for cattles. A practical volume forinjection of a vaccine is 1 ml for small animals and 2 to 5 or 10 ml forbig animals. As CMC (the critical micelle concentration) for Quil A is0.03%, 1 ml will imply an amount of 300 μg when 1 ml is injected. Afterinjection, however, due to the dilution effect, the concentration willbecome lower than CMC and the micelle will become unstable.

According to the present invention, however, the saponin and especiallythe Quil A molecules will be bounded together with cholesterol moleculesso that a relatively stable complex is formed at very lowconcentrations. This complex is effective as adjuvant in a dose, whichcorresponds to 0.1 μg Quil A, i.e. 100 times lower than when Quil A ispresented in free form.

BRIEF DESCRIPTION OF THE DRAWINGS

The Figures show:

FIG. 1 shows an electron microscope picture of a typical matrix:

FIG. 2 shows U.V. eluation profiles for subfractions of Quil A:

FIG. 3 demonstrates HPTLC-separation of Quil A and its subfractions:

FIG. 4A shows the FAB-mass-spectrum for the new substance B2 accordingto the invention;

FIG. 4B shows the FAB-mass-spectrum for the new substance B3 accordingto the invention;

FIG. 4C shows the FAB-mass-spectrum for the new substance B4B accordingto the invention;

FIG. 5A shows the ¹³ C-NMR-spectrum in a first region, for the newsubstance B2;

FIG. 5B shows the ¹³ C-NMR-spectrum in a first region, for the newsubstance B3;

FIG. 5C shows the ¹³ C-NMR-spectrum in a first region, for the newsubstance B4B;

FIG. 6A shows the ¹³ C-NMR-spectrum in a second region, for the newsubstance B2;

FIG. 6B shows the ¹³ C-NMR-spectrum in a second region, for the newsubstance B3;

FIG. 6C shows the ¹³ C-NMR-spectrum in a second region, for the newsubstance B4B;

FIG. 7 shows the complete ¹³ C-NMR-spectrum for the substance B2;

FIG. 8 shows the complete ¹³ C-NMR-spectrum for the substance B3;

FIG. 9 shows the complete ¹³ C-NMR-spectrum for the substance B4B;

FIG. 11A shows the ¹ H NMR-spectrum in a first region for the newsubstance B2;

FIG. 11B shows the ¹ H NMR-spectrum in a first region for the newsubstance B3;

FIG. 11C shows the ¹ H NMR-spectrum in a first region for the newsubstance B4B;

FIG. 12A shows the ¹ H NMR-spectrum in a second region for the newsubstance B2;

FIG. 12B shows the ¹ H NMR-spectrum in a second region for the newsubstance B3;

FIG. 12C shows the ¹ H NMR-spectrum in a second region for the newsubstance B4B;

FIG. 13 shows parts of the spectra in FIGS. 7 and 8; and

FIG. 14 shows a 2-dimensional E-spectrum for substance B3.

The invention will now be described further with the following example.

EXAMPLE 1

Matrix (Cholesterol-Quil A complex)

1 mg of cholesterol dissolved in 20% MEGA-10 (in H₂ O) was mixed with 5mg of solid Quil A. The Quil A was dissolved and the mixture wasdialysed extensively against PBS, first over night at room temperature,then at +4° C. The iscom matrixes were purified from excess Quil A andcholesterol by pelleting through 30% (w/w) sucrose (TST 41.13 rotor 18h, 39.000 rpm, 10° C.). The pelleted matrixes were dissolved in PBS andthe concentration adjusted to 1 mg/ml (traced by a small amount of H³-cholesterol).

EXAMPLE 2

MDP (muramyldipeptide, Sigma, adjuvant peptide) was conjugated tophosphatidyl ethanolamine (PEA) using N-ethyl-N'-(dimethylaminopropyl)carbodiimide hydrochloride as described by Lefrancier et al., 1977(Lefrancier, P., Choay, J., Derrien, M. and Lederman, I. (1977) Int. J.peptide Protein Res. 9:249-257).

To 1 mg of cholesterol (in 20% MEGA-10 in H₂ O) was added an equimolaramount of MDP-PEA (in MEGA-10 or DMSO or any other water misciblesolvent), an equimolar amount of phosphatidyl choline and 7 mg of Quil A(a slight excess in comparison to 5 mg that is required forIM-formation). After a short incubation at room temperature (15-30 min)the mixture was extensively dialysed against PBS (room temperature 4-12h, then at +4° C.).

After completed dialysis, the matrix-complexes with the additionaladjuvant integrated were purified from excess Quil A by pelletingthrough 10% sucrose.

EXAMPLE 4

To 1 mg of cholesterol (in 20% MEGA-10 in H₂ O). was added an equimolaramount of Avridine(N,N-dioctadecyl-N'N'-bis(2-hydroxyethyl)propenediamine (in MEGA-10 orDMSO or any other water micible solvent), an equimolar amount ofphosphatidyl choline and 7 mg of Quil A (a slight excess in comparisonto 5 mg that is required for IM-formation). After a short incubation atroom temperature (15-30 min) the mixture was extensively dialysedagainst PBS (room temperature 4-12 h, then at +4° C.).

After completed dialysis, the matrix-complexes with the additionaladjuvant integrated were purified from excess Quil A and adjuvant bypelleting through 10% sucrose (the same method as described on page 14,last paragraph).

EXAMPLE 4

To 1 mg of cholesterol (in 20% MEGA-10 in H₂ O) was added an equimolaramount of DDA (dimethyl dioctadecyl ammonium bromide (in MEGA-10 or DMSOor any other water micible solvent), an equimolar amount of phosphatidylcholine and 7 mg of Quil A (a slight excess in comparison to 5 mg thatis required for matrix-formation). After a short incubation at roomtemperature (15-30 min) the mixture was extensively dialysed against PBS(room temperature 4-12 h, then at +4° C.).

After completed dialysis, the matrix-complexes with the additionaladjuvant integrated were purified from excess Quil A and adjuvant bypelleting through 10% sucrose (the same method as described on page 14,last paragraph).

EXAMPLE 5

2 g of Mega 10 is added to 10 ml of water before the addition of 200 mgcholesterol, and the cholesterol is dispersed byultrasonication/ultraturrax. As much as 1.6 ml of this mixture can beadded to the 10 ml of 2% Quil A-solution. The reaction mixture clarifiescompletely after less than one hour indicating that all the cholesterolhas been reacted. It can be seen in the electron microscope that theconcentration of matrix is very high even if the concentration ofdetergent in this case is 10%. Removal of the detergent by dialysis orultrafiltration does not quantitatively affect the number of matrixparticles, and the solution of matrix strays completely clear.

This experiment indicates that matrix formation takes place when thesurfactants are present in the reaction mixtures, and that completematrix formation takes place in very high concentrations of detergent.

EXAMPLE 6

Preparation of the Quil A components B2, B3 and B4B according to theinvention.

5 g Cortex quillajae (Nordiske Droge of Kemikalieforretning, Copenhagen,Batch nr 8372) and 50 ml destillated water was mixed by a magneticaIstirrer for 3 hours at room temperature. The liquid phase was separatedthrough a Buchner funnel by a filter paper and was purified by filteringthrough a Metricel Gelman membrane 0.22μ. Such an extract contains 2.5%dry material.

The crude extract was dialysed against 200 volumes of destillated waterin a Visking-tube without weld 20/32 for 48 hours with exchange of waterafter 24 hours. This extract is called DQ.

The dialysed extract above was subjected to ion exchange chromatography.A column of DEAE-cellulose equilibrated with 0.1M Tris-HCl pH 7.5 wasprepared (Whatman DE52) in a K 9/15 column (Pharmacia Fine Chemicals).The bed material was equilibrated with 0.1M Tris-HCl buffer pH 7.5. Thecolumn was eluted either stepwise or by a linear salt gradient at a flowrate of 60 ml/h using a peristaltic pump. 50 ml DQ was introduced on thecolumn and 300 drop (equivalent to approx. 5 ml) fractions werecollected. Under these conditions, some of the substances in DQ passedunbound through the column, as will be seen from FIG. 2A (peak A).Elution was continued until no UV absorption was detectable. Theabsorption of the effluent liquid was recorded at 280 nm by a Uvicord IIsystem (LKB-Produkter), and fractions were collected by a GoldenRetriever (ISCO). At this point a buffer containing 0.2M NaCl made up instart buffer was introduced. As can be seen in FIG. 2A, a peak B iseluted. However, some substances were still attached to the bed materialto such a degree that elution was difficult even with concentrated NaCl.These substances were the ones that contributed to the brownish colourof DQ, whereas peak A and B were only slightly coloured or completelycolourless, respectively. In the next purification step, peak B waspooled and subjected to gel exclusion chromatography on Sephadex G50fine equilibrated with M/50 phosphate buffer pH 7.5 in a K 16/70 columneluted at a flow rate of 10 ml/h. Desalting was carried out on SephadexG25 medium in a K 16/40 column. Elution was carried out using ahydrostatic head of 50 cm. As will be seen from FIG. 2B, the UV profileshowed 3 peaks. Peak C was eluted in the void volume (as determined byBlue Dextran 2000, Pharmacia Fine Chemicals) and peak E was eluted inthe total volume of the column (also determined by potassium chromate).Peak D was well separated from peak C and E, but as can be seen in FIG.2B, the presence of a shoulder indicated that peak D consisted of atleast two substances.

Consequently, peak D was pooled and subjected to a new separation onDEAE-cellulose. The starting conditions were the same as in the firstion exchange experiment, and as a result all the material was adsorbedto the column. Elution was now continued with a linear NaCl gradientincreasing from 0 to 1 molar (made up in the start buffer) in the courseof 300 ml. The result of this experiment is shown in FIG. 2C. Two peaksF and G appeared in the UV profile. F was clearly separated and a singlesubstance (section 3.3), but peak G could not be isolated since it wascontaminated with F. In order to investigate the homogeneity of peak F,it was pooled, desalted on Sephadex G25, and rechromatographed in anidentical experiment. As can be seen from FIG. 2D, only one symmetricalpeak (H) appeared at the position of peak F.

Anyone of the fractions (also the DQ-extract) can be further purified asfollows.

Fraction H was lyophilized and dissolved in chloroform/methanol/H₂ O(60:40:9, v/v/v). 200 mg was then applied to an HPLC column (4.5×50 cm)packed with silicic acid, Iatrobeads RS-8060 (Iatron Labs, Tokyo,Japan). A pump speed of 5 ml/min was used for pumping a total of 2 Lsolvent, collecting 200 fractions of 10 ml with a gradient ofchloroform/methanol/H₂ O (60:40:9 to 50:40:10) The fractions wereanalyzed with thin layer chromatography in the following way. 2 μl ofevery second fraction was analyzed by developing thin layer liquidchromatography plates (TLC-plates) (HPTLC, Merck, Bodman Chemicals,Gibbstown, N.J.) in chloroform/methanol/0.2% CaCl₂ (50:40:10 v/v/v) andthe glycosides were determined by being greencoloured with anisaldehydereagent (acetic acid/sulphoric acid/paraanisaldehyde (98:2:1)). The QuilA starting material was used as a reference of the R_(f) -value. (SeeFIG. 3, which shows a HPTLC-separation of: lane 1, Quil A (fraction H);lane 2, B1; lane 3, B2; lane 4: B3: lane 5, B4A; and lane 6, B4B).

Fractions that comigrate with B2, B3 and B4B having identical R_(f)-values were pooled and analyzed for purity with TLC. These crudefractions usually must be chromatographed two times in order to becomepure enough. Fractions B1 and B4A are inactive and therefore are notseparated further.

The thus enriched components were further purified on an HPLC column(21.2×250 mm) packed with 5μ spherical silica particles (Zorbax Si,DuPont, Wilmington, Del.). 40 mg enriched fraction B2, B3 or B4Bdissolved in 1 ml chloroform/methanol/H₂ O (60;40;9 v/v/v) was put onthe column. A pump speed of 3 ml/min was used for pumping a total of 0.9l solvent, collecting 300 fractions of 3 ml with a gradient ofchloroform/methanol/H₂ O (60;40;9 to 50;40;10 v/v/v). Fractions wereanalyzed on glass-backed HPLTC-plates as above. Purified fractions werepooled and evaporated to dryness in a rotary evaporator <30° C.,dessiccated and stored in <-20° C. Approximately 20-25 rounds of thispurification step was used i.e. using (20-25)×200 mg=4-5 g Quil Astarting material, including rechromatography to prepare 1 g of fractionB3. The yield of B2 and B4B was about 40% of the yield of B3.

The so prepared components B2, B3 and B4B were analyzed as follows.

a) Mass Spectrometry

Negative FAB-MS, FIG. 4, and positive FAB-MS (data not shown) werecarried out for determination of molecular weights of the purified QuilA components B2, B3, B4A, and B4B. The data shown in FIG. 4 arepreliminary and will have to be reacquired in a neutral pH matrix suchas glycerol rather than in triethanolamine which was used for thespectra shown in FIG. 4. This is necessary because extremealkali-lability of the compounds, pH>8.5 have been demonstrated. Peaksat m/z 595, 744, and 893 stem from the matrix triethanolamine and shouldbe disregarded. Our fraction B4A, which does not have any adjuvant orISCOM particle forming capacity, seem to be identical with thatdescribed by Komori et al (for structure, see FIG. 10). The peakscorresponding to molecular weights of the three thus far mostinteresting glycosides are at: m/z 1988, B2; m/z 2150, B3; and m/z 1862,B4B.

b) ¹³ C-NMR

FIGS. 5 and 6 show two regions, aliphatic carbon (8-45 ppm) and anomericcarbon (90-115 ppm), respectively, of the ¹³ C-NMR spectra for the fullsize fractions: A, B2 (20 mg) B, B3 (80 mg); and C, B4B (40 mg). Allspectra were obtained in the solvent-system, chloroform/methanol/water(30:60:8, v/v/v). The triterpenoid region is well resolved (8-45 ppm,FIG. 6) and has been partially assigned as seen in Table 4.

                  TABLE 4                                                         ______________________________________                                        Partial .sup.13 C-NMR signal assignment (ppm) for β-amyrin               five-ring segment of fractions B2, B3, and B4B (see FIG. 5)                   obtained in chloroform/methanol/water (30:60:8, v/v/v).                       Carbon#   B2     B3         B4B  Reference                                    ______________________________________                                        C9.sup.a                                                                      b                                                                             b                                45.5                                         C10       36.4   36.2       36.3 37.0                                         C12       122.5  122.5      122.5                                                                              123.1                                        C13       144.1  146.6      143.6                                                                              144.8                                        C14       41.5   41.8       41.9 41.6                                         C15       30.0   30.5       30.7 30.7                                         C18       43.0   42.8       41.9 42.7                                         C20       30.7   30.6       30.6 30.7                                         C25       16.0   16.1       16.0 15.9                                         C26       17.7   17.3       17.6 17.6                                         C29       32.9   32.9       32.9 32.2                                         ______________________________________                                         .sup.a Numbered as in FIG. 5.                                                 .sup.b Hidden under methanol signal of solvent (confirmed with a DEPT         experiment).                                                             

These assignments have been performed from studying a large number ofreference-spectra obtained in various solvents and by analyzing thesignals that are solvent-independent by a statistical comparison (datanot shown). FIG. 6 shows the region between 80-148 ppm in the spectra ofthe three compounds, A, B2; B, B3; and C, B4B, featuring two double-bondcarbon signals at 122 and 143 ppm corresponding to C-12 and C-13,respectively, in the β-amyrin skeleton (FIG. 10). The atomeric-carbonregion, between 90-115 ppm, shows the presence of approximately 9-10signals corresponding to the same amount of sugar-residues in thecompounds.

Conclusion: Structural differences can be identified between fractions:A, B2; B, B3; and C, B4B, in both spectral regions corresponding tomainly the triterpenoid region and the oligosaccharide portions of themolecules, respectively. The exact amounts of sugars can not bedetermined at this point.

c) ¹ H NMR

FIG. 11 demonstrates the full proton spectrum (0-10 ppm) and FIG. 12partial proton spectrum (anomeric region, 4.0-6.0 ppm), respectively, offractions: A, B2 B. B3: and C, B4B. The spectra are obtained fromsamples (≈10 mg, ≈600 scans) dissolved in DMBO.d₆ /D₂ O (98:2, v/v). Tothe far left in the spectrum (FIG. 11), at 9.4 ppm, the signal from thealdehyde proton on carbon-24 (see FIG. 5) is found. The doublet natureof this peak, a peak which is supposed to be a singlet, since it has noneighbouring protons to couple to, offers an explanation to theunusually complex anomeric region which is poorly resolved (as seen inthe expansion in FIG. 12).

The doublet can be due to an aldehyde proton in two different compoundsor to the presence of chemical exchange between two differentpopulations of the same molecule (this process is slow enough in NMRtime scale to be observed) thus explaining the different integrals ofthe peaks in the doublet at different temperatures as shown in FIG. 13and Table 5.

                  TABLE 5                                                         ______________________________________                                        Temperature                Difference                                                                            Integral Quote                             in Degree K.                                                                           Shift 1 Shift 2   in Shifts                                                                             Shift 1/Shift 2                            ______________________________________                                        301      9.46    9.44      0.02    1.61                                       351      9.47    9.46      0.01    1.99                                       361      9.48    9.47      0.01    2.23                                       ______________________________________                                    

FIG. 13 and Table 5 (above) demonstrate that the relative integral ofthe peaks varies with temperature and that the two peaks move closer toeach other at a higher temperature, both indicating that it can not betwo different molecules but rather two different populations of the samemolecule. This would explain the complex anomeric region by suggestingthat many atomeric protons in the molecule would have double resonancesdue to different chemical environments in the two populations. However,the present set of data indicates that differences in the glycosylationof the compounds could provide part of the explanation of theirstructural differences (FIG. 12), by demonstrating different amount ofanomeric proton signals in the spectra. The FAB-MS data for fractionsB2, B3 and B4B also does not rule out the formal possibility that twosimilar size molecules, with very similar physico-chemical properties,exist that have the same amount of sugars but differ inlinkage-positions and/or sequence.

Conclusion: In general, 1-dimensional ¹ H NMR spectra from 8-10 sugarcontaining earlier unknown molecules are not sufficient for assignmentof protons and detailed structural characterization. For resolving allsignals and for making proper assignments through out the compounds itwill be necessary to use the 2-dimensional NMR technique as well aschemically degrade the compounds for analysis. Both homonuclear (¹ H-¹H) and heteronuclear (¹ H-¹³ H) COSY, TOCSY as well as NOESY. The2-dimensional proton phase sensitive correlation double quantum filteredNMR spectrum (DQFPSCOSY) for fraction B3 is shown in FIG. 14.

d) Summary of Structural Data

The conclusion of data generated thus far is that the active fractionsthat have adjuvant activity and ISCOM particle forming capacity in QuilA contain unique glycosylated triterpenoid-saponins that differ betweeneach other in both their triterpenoid and glycan parts. They have anapproximate structure like the one described in FIG. 10 and consist of afive-ring steroid skeleton of β-amyrin type and contains 8-11 sugarresidues.

EXAMPLE 7

0.1 mg cholesterol was mixed with ³ H-cholesterol (10 mg/ml dissolved in20% MEGA-10 in H₂ O) and 0.5 mg B2 or B3 or B4B or mixtures thereof. Thevolume was adjusted to 0.5 ml and the mixture dialysed against PBS on apreparation treated with ammonium molybdate (negative colouringtechnique). The dialysed preparations were analysed for the presence ofcomplex with iscom structure by electron microscopy (EM) and analyticalgradient centrifugation. In EM the iscom structure is characterized by acage-like particle with a diameter of 40 nm composed of subunits withannular structure with a diameter of 12 nm. For sedimentation studiesthe sample is placed over a sacharose gradient (10-50%) and centrifugedfor 18 hours, +10° C. in a TST 41,14 rotor, 40 000 rpm. The gradient iscollected in 18 fractions (fraction 1=the bottom and fraction 18=thetop). By localizing the 3H-cholesterol activity in the gradient, one cantell the sedimentation constant and see if complexes have been made. B4Bforms typical iscom structures with cholesterol but has no potentadjuvant activity.

B2 does not form iscom-like structures with cholesterol but binds tocholesterol. Together with B4B, B2 forms iscom-like structures withcholesterol. B2 has a weak adjuvant activity.

B3 binds to cholesterol but not in iscom-like structures. With B4B, B3like B2, can form iscom-like structures with cholesterol. B3 hasadjuvant activity.

We claim:
 1. A vaccine comprising an immunomodulating agent having aniscom-like structure and comprising within said iscom-like structure atleast one lipid and at least one saponin, said iscom-like structurebeing free of incorporated antigens; one or more antigens in admixturewith said immunomodulating agent but not integrated into said iscom-likestructure; and a pharmaceutically acceptable vehicle.
 2. A kit for humanor veterinary medical use, comprising: (a) an immunomodulating agenthaving an iscom-like structure and comprising within said iscom-likestructure at least one lipid and at least one saponin, said iscom-likestructure being free of incorporated antigens; and (b) one or moreimmunomodulating substances and a pharmaceutically acceptable vehicle;said components (a) and (b) being confined in separate containers.
 3. Amethod of inducing an immunomodulatory response in a patient in needthereof, comprising administering (a) an antigenically effective amountof at least one antigen, and (b) an immunomodulating agent in an amounteffective to produce an immunomodulatory effect on the action of said atleast one antigen, said immunomodulating agent having an iscom-likestructure and comprising within said iscom-like structure at least onelipid and at least one saponin, said iscom-like structure being free ofincorporated antigens; said components (a) and (b) being administered inadmixture, or separately.
 4. The method according to claim 3, whereinsaid at least one antigen is in multimeric form.
 5. The method accordingto claim 3, wherein said at least one lipid is a sterol.
 6. The methodaccording to claim 5, wherein said sterol is cholesterol.
 7. The methodaccording to claim 3, wherein said at least one saponin is atriterpensaponin.
 8. The method according to claim 7, wherein saidtriterpensaponin is Quil A.
 9. A kit for human or veterinary medicaluse, comprising: (a) an immunomodulating agent having an iscom-likestructure and comprising within said iscom-like structure at least onelipid and at least one saponin, said iscom-like structure being free ofincorporated antigens; and (b) one or more immunomodulating substancesand a pharmaceutically acceptable vehicle; said components (a) and (b)being in admixture in a single container.
 10. The vaccine according toclaim 16, wherein said saponin is isolated from Quillaja SaponariaMolina of β-amyrin type with 8-11 carbohydrate moieties, and is selectedfrom the group consisting of:a) substance B2 having a molecular weightof 1988, a carbon 13 NMR spectrum as shown in FIGS. 5A and 6A, and aproton NMR spectrum as shown in FIGS. 11A and 12A; b) substance B3having a molecular weight of 2150 and a carbon 13 NMR spectrum as shownin FIGS. 5B and 6B, and a proton NMR spectrum as shown in FIGS. 11B and12B; and c) substance B4B having a molecular weight of 1862, a carbon 13NMR spectrum as shown in FIGS. 5C and 6C, and a proton NMR structure asshown in FIGS. 11C and 12C.